Keratins are a class of structural proteins widely represented in biological structures, especially in epithelial tissues of higher vertebrates. Keratins may be divided into two major classes, the soft keratins (occurring in skin and a few other tissues) and the hard keratins (forming the material of nails, claws, hair, horn, feathers and scales).
The toughness and insolubility of hard keratins, which allow them to perform a fundamental structural role in many biological systems, are the desirable characteristics found in many of the industrial and consumer materials derived from synthetic polymers. In addition to possessing excellent physical properties, keratin, as a protein, is a polymer with a high degree of chemical functionality and consequently exhibits many properties that synthetic polymers cannot achieve. Keratin is therefore, well suited to the development of medical products with high-value, niche market applications. Medical materials which are absorbed (resorbed) by the body tissues after fulfilling their function are an example of an area of high value products in which the specific characteristics of keratin allow it to outperform both natural and synthetic competitive materials.
Yamauchi (K. Yamauchi, M. Maniwa and T. Mori, Journal of Biomaterial Science, Polymer edition, 3, 259, 1998) demonstrate that keratins can be processed into matrices that are considered biocompatible by virtue of their in vitro and in vivo properties. The processing methods used to make these materials require large concentrations of reducing agents, such as thiols, and processing conditions that are not suitable for commercial production of materials.
Kelly (WO03/19673) shows that keratins can be processed into complex shapes using commercially viable chemistries and processing conditions.
Blanchard (U.S. Pat. No. 5,358,935 and US2003/0035820A1) demonstrates that keratins can be extracted from human hair using high concentrations of reductants, or harsh oxidants, and processed to produce materials useful in some soft tissue applications. However, extraction and reconstitution methods are harsh and cause degradation to the keratin, through irreversible oxidation of the distinctive keratin amino acid cystine to cysteic acid, or through exposure of the protein to high pH conditions that lead to peptide hydrolysis. This results in many beneficial characteristics of the protein being lost, in particular the toughness necessary for hard tissue applications.
In order to produce keratin biomaterials suitable for orthopaedic applications, methods of processing are needed that maintain the keratin characteristics and provide materials with good toughness properties. This invention describes such materials and their methods of production.
U.S. Pat. No. 6,432,435 claims a tissue engineering scaffold having a keratin with hydrophilic groups, the keratin being bound with keratin-keratin disulfide bonds. The patent however provides no disclosure as to how a sulfonated keratin can be incorporated into a hard tissue such as bone. The examples provided all relate to its use in soft tissue or porous structures.
The present inventors have found that keratin can be incorporated into hard tissue such as bone and hence used in the treatment of bone injury.
Many tissues of the body including bone are continually renewed. New bone matrix (which will become mineralized) is laid down principally by specific cells called osteoblasts, and the different components of bone are removed by osteoclasts. An implanted material which is removed and replaced with bone tissue by this biological process will have a greater advantage over those materials which break down by other mechanisms within the body e.g. chemical degradation. It is desirable that new bone is formed juxtaposed to the surface of the implanted material, thereby integrating this material into the tissue until it is completely resorbed and replaced.
Bone may be categorized into four microstructural components: cells, organic matrix, inorganic matrix, and soluble signalling factors. Osteoblasts are metabolically active secretory cells that express soluble signalling factors and osteoid, a product whose extracellular modification yields an organic insoluble substratum consisting mostly of type I collagen. Expression of these products by osteoblasts occurs during maintenance (e.g. remodelling), and repair of bone. Monocyte-macrophage precursors found in the bone marrow enter the circulation, and through asynchronous fusion produce a multinucleated cell up to 100 microns in diameter with an average of 10 to 12 nuclei, known as an osteoclast. Osteoclasts have a ruffled border and this constitutes the resorptive territory of the osteoclast where enzymatic breakdown of the bone surface occurs. The term ‘remodelling’ is used to describe the dynamic events associated with bone repair and homeostasis in the mature individual. The sum of the processes associated with homeostatic remodelling is known as activation-resorption-formation. Osteoblasts are activated by signalling factors and vacate an area of bone; osteoclasts become stimulated, home in to the osteoblast-vacant zone, attach, resorb, and, in response to an as yet unidentified signal, cease resorbing and abandon their attachment. Osteoclastic resorptive pits become repopulated by a contingent of osteoblasts that express osteoid, which calcifies, restoring bone. In humans, the activation-resorption-formation processes take between 3 and 6 months.
Following an insult to bone (e.g. fracture or surgical removal of a tumor) there is extensive bleeding and in 2 to 5 days the haemorrhage forms a large blood clot. Neovascularization begins to occur peripheral to this blood clot. There is also the standard inflammatory response occurring in the surrounding soft tissues leading to polymorphonuclear leucocytes, macrophages, and mononuclear cells accumulating in the periphery of the clot. By the end of the first week, most of the clot is organised by invasion of blood vessels and early fibrosis. The earliest bone (woven bone) is formed after 7 days. Since bone formation requires a good blood supply, the woven bone spicules begin to form at the periphery of the clot where vascularisation is greatest. Pluripotential mesenchymal cells from the surrounding soft tissues and from within the bone marrow give rise to osteoblasts that synthesize the woven bone. Frequently cartilage is also formed and eventually is replaced by endochondral ossification. The granulation tissue containing bone-cartilage is termed a callus (Inflammatory phase).
After the first week, the next stage begins and extends for several months, depending upon the degree of movement and fixation. By this stage, the acute inflammatory cells have dissipated and the reparative process involving the differentiation of pluripotential cells into fibroblasts and osteoblasts commences. Repair proceeds from the periphery towards the centre and accomplishes two objectives: one, it organises and resorbs the blood clot; and two, more importantly, it furnishes neovascularisation for the construction of the callus, which eventually bridges the bone-deficient site. The events leading to the repair are as follows. Large numbers of osteoclasts from the surrounding bone move into the healing site. New blood vessels accompany these cells supplying nutrients and providing more pluripotential cells for cell renewal. The site is remodelled by osteoclasts (Reparative phase).
In several weeks the callus has sealed the bone ends and remodelling begins, in which the bone is reorganised so that the original cortex is restored (Remodelling phase).